1. Field of the Invention
The present invention relates to a focal position determining method and a focal position determining apparatus that determine a focal position of an objective lens focused on an observed target region in a specimen.
The present invention relates to a method and an apparatus for observing or measuring a test specimen of a biological origin that emits a feeble light with magnification imaging optical unit including a lens.
2. Description of the Related Art
[1] In recent years, an imaging technique of a biological sample utilizing fluorescence has taken a great role for a research of a bioscience. A specific protein is marked, and light emission is utilized, whereby various life phenomena happening inside or outside a cell can be observed. Further, dynamic actions of various life phenomena can be known in real time. In recent days, in particular, the use of a fluorescent protein such as GFP (Green Fluorescent Protein) makes it possible to stably and easily realize an imaging of a structure in a cell, so that various life phenomena has been steadily unraveled.
Further, a luminescence-related gene that expresses a bioluminescent protein (specifically, luciferase, aequolin, or the like) has frequently been utilized for various function analyses in a cell (specifically, a luminescence-related gene has frequently been utilized as a reporter molecule of a protein expression). In performing the function analyses described above, it is extremely significant to describe a clear image by bringing a lens into focus on a specific region in a cell, or to efficiently receive light emitted from a specific bioluminescent protein transduced to a cell. However, since an intensity of light of self-luminescence from a biological specimen is generally extremely feeble, the luminescence from the biological specimen cannot directly be confirmed with naked eyes in most cases. Even when an optical element (e.g., detection lens, etc.) is adjusted to a specimen emitting a feeble light, it is naturally difficult to bring a lens into focus on the specific region in the specimen with naked eyes.
In view of this, various methods for focusing light on a specimen in a specimen container have been disclosed. However, all of the methods are applicable to the case in which the light from a specimen can be confirmed (visually) with naked eyes. For example, JP-T-2002-541430 and JP-T-2002-542480 disclose a method in which a position of a bottom surface of a specimen container is detected, and light is focused on a specimen in the specimen container based on the detected positional information. In JP-T 2002-541430, light is irradiated to the bottom surface of the specimen container through an objective lens, the intensity of the mirror-reflected light from the bottom surface of the specimen container is sequentially detected while moving the light irradiation position vertically, the position of the bottom surface of the specimen container is detected based on the detected intensity of light, the position of the specimen in the specimen container is estimated based upon the detected positional information, and light is focused on the estimated position. In JP-T-2002-542480, light is diagonally irradiated to the bottom surface of the specimen container from below through an objective lens, a deviation amount of the mirror-reflected light from the bottom surface of the specimen container on an XY plane is detected by a photodetector, the position of the bottom surface of the specimen container on an optical axis is detected based upon the detected deviation amount, the position of the specimen in the specimen container is estimated based upon the detected positional information, and light is focused on the estimated position. Thus, the focal position of the objective lens can be focused on the specimen in the specimen container.
JP-A-2004-354650 and JP-A-2005-173288 disclose a method in which, with the use of a microscope for observing a phase object such as a cell with a bright field, the position of an objective lens is shifted in the forward or rearward direction from the general focusing position to be fixed to thereby obtain an observation image with a high contrast by defocus, in order to observe a cell. With this method, the observation image of the cell, which is the phase object, can be obtained with a high contrast.
[II] In recent years, a technique for analyzing a specimen of a biological origin by utilizing fluorescence or luminescence (chemiluminescence and bioluminescence) has taken a great role for a research of a bioscience. A specific protein is marked, and fluorescence or luminescence is utilized, whereby various life phenomena happening inside or outside a cell can actually be measured. Further, dynamic actions of various life phenomena can be found in real time. In recent days, in particular, the use of a fluorescent protein such as GFP (Green Fluorescent Protein) makes it possible to stably and easily realize an imaging of a structure in a cell, so that the analyzing technique by means of a fluorescent image has steadily been contributed to the unraveling of various life phenomena.
In the analysis of a specimen of a biological origin by means of a fluorescent image, an observation image is optically or electrically (digitally) magnified, and then, an image is analyzed, when a specimen is too small. As one example of this technique, there has been disclosed an apparatus that counts a number of microorganisms by using a magnified image obtained by magnifying a fluorescent image, which is obtained by dyeing the proliferated microorganisms with a fluorescent pigment (see JP-A-2005-172680).